EP1129215B1 - Ligationsassemblierung und detektion von polynucleotiden auf einem festträger - Google Patents

Ligationsassemblierung und detektion von polynucleotiden auf einem festträger Download PDF

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EP1129215B1
EP1129215B1 EP99945166A EP99945166A EP1129215B1 EP 1129215 B1 EP1129215 B1 EP 1129215B1 EP 99945166 A EP99945166 A EP 99945166A EP 99945166 A EP99945166 A EP 99945166A EP 1129215 B1 EP1129215 B1 EP 1129215B1
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immobilized
polynucleotide
oligonucleotides
assembly
support
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EP1129215A1 (de
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Michael W. Hunkapiller
Andrew C. Hiatt
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HIATT, ANDREW C.
Applied Biosystems Inc
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Applera Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates generally to methods for assembly, analysis, detection, and cleavage of polynucleotides on a solid-support by annealing, ligation, and extension steps.
  • the classic method of de novo gene synthesis entails sequential annealing (hybridization) and ligation of the component synthetic oligonucleotides, a few at a time, in a homogeneous aqueous solution (Khorana, 1979; Blackburn, 1996).
  • a mixture of overlapping, complementary oligonucleotides are annealed under conditions that favor formation of a correct double-stranded fragment (duplex DNA) with strand interruptions (nicks) at adjacent positions along the two strands.
  • the resultant construct is then isolated and submitted to subsequent rounds of annealing, ligation, and isolation.
  • WO 90/00626 discloses a method of polynucleotide assembly in which a series of overlapping oligonucleotides are annealed, extended if necessary to fill in gaps, and ligated.
  • Limitations to the classic method of gene synthesis include the known imperfections in chemical oligonucleotide synthesis, especially long oligonucleotides, resulting in impurities resulting from (i) failed-to-couple, truncated sequences, (ii) nucleobase-modified sequences, (iii) incompletely deprotected sequences, and (iv) other nucleotidic and non-nucleotidic by-products. Hybridization of impure oligonucleotide mixtures can lead to mismatches and impaired hybridization and ligation efficiency. The net result is low yields of functional, correct sequence oligonucleotides for use in synthetic gene assembly. An efficient method for the rapid and economical assembly of polynucleotides, i.e. genes or gene fragments, is desirable.
  • the present invention is directed towards novel methods for assembly and detection of a polynucleotide on a solid-support.
  • the methods are directed to rapid, efficient, low-cost, and large-scale synthesis of polynucleotides for use, for example, as synthetic genes for recombinant protein expression, as probes for diagnostic assays, or antisense therapeutic agents.
  • the resulting polynucleotides on solid-support can be (i) amplified by the polymerase chain reaction (PCR), (ii) quantitated and detected by fluorescence-based, hybridization and exonuclease assays, (iii) manipulated for useful purposes while attached to the solid-support, or (iv) cleaved from the solid-support.
  • PCR polymerase chain reaction
  • the present invention comprises a method of synthesis of a polynucleotide on a solid-support where the method includes steps of annealing oligonucleotides to an immobilized oligonucleotide on a solid-support, ligating nick sites repeating the annealing and ligating 1-100 cycles, removing the bridging oligonucleotides under denaturing conditions, annealing a primer to the immobilized ligation product and extending portions of the polynucleotide with polymerase and nucleotide 5' triphosphate to generate double-stranded polynucleotides on a solid-support.
  • the first end of an oligonucleotide is immobilized on a solid-support.
  • the non-immobilized end bears a phosphate group.
  • One or more bridging oligonucleotides comprising 6-40 nucleotides and forming gaps of one or more nucleotides in the non-immobilized strand and two or more assembly oligonucleotides comprising 20-200 nucleotides are annealed to the immobilized oligonucleotide such that a ligatable nick is formed between adjacent assembly oligonucleotides.
  • the nick sites are ligated thereby forming an immobilized ligation product.
  • a primer is annealed to the immobilized ligation product and extended to create a double-stranded polynucleotide. The annealing and ligation steps are repeated enough times to assemble the designed, immobilized double-stranded polynucleotide.
  • Various combinations of assembly and bridging oligonucleotides for the assembly of polynucleotides are illustrated in Figures 1-6.
  • a bridging oligonucleotide comprising 6-40 nucleotides anneals to the immobilized oligonucleotide, whereby the bridging oligonucleotide is complementary to the non-immobilized end of the immobilized oligonucleotide and creates a first double-stranded fragment with an overhang.
  • An assembly oligonucleotide comprising 20-200 nucleotides typically longer than the bridging oligonucleotide and complementary to the overhang of the non-immobilized strand, anneals at a nucleotide adjacent to the non-immobilized end of the immobilized oligonucleotide to create a second double-stranded, fragment having a nick site and an overhang. Additional assembly and bridging oligonucleotides are introduced and anneal to form nicks in the immobilized strand and gaps in the non-immobilized strand. The nick sites are ligated in the immobilized strand by DNA ligase or by chemical ligation means.
  • the annealing and ligation steps are repeated 1-100 cycles and the bridging oligonucleotides are removed under denaturing conditions.
  • the non-immobilized strand is extended by polymerase, and primer, and nucleotide 5' triphosphates to create a double-stranded polynucleotide.
  • a preferred aspect of the present invention provides methods to detect and quantitate the assembled, double-stranded polynucleotide on the solid-support by fluorescent hybridization assay.
  • the method further comprises annealing a self-quenching, fluorescence probe including reporter and quencher moieties and complementary to said polynucleotide after synthesis is completed.
  • the probe may be comprised of nucleotides near the 5' terminus which are substantially complementary to the nucleotides near the 3' terminus whereby the unannealed probe exists in a quenched state.
  • the quenching effect is lost or substantially minimized and fluorescence can be detected.
  • methods of the invention can be used to amplify the double-stranded polynucleotide on a solid-support by the polymerase chain reaction (PCR) or to detect and quantitate the product of the polymerase chain reaction by the fluorescence based, exonuclease assay (Lee, 1993; Holland, 1991).
  • PCR polymerase chain reaction
  • the methods of the present invention can be modified to cleave the immobilized single-stranded polynucleotide from the solid-phase into solution by chemical or restriction enzyme cutting.
  • a device can be constructed to synthesize a polynucleotide on a solid-support by ' automating the steps of annealing, ligation, and primer extension in a cyclical manner.
  • Liquid reagents can be delivered from vessels to the solid-supports under microprocessor control according to a program.
  • solid-support refers to a material in the solid-phase that interacts with reagents in the liquid phase by heterogeneous reactions.
  • Solid-supports can be derivatized with oligonucleotides by covalent or non-covalent bonding through one or more attachment sites, thereby "immobilizing" an oligonucleotide to the solid-support.
  • annealing is used synonymously with “hybridization” and refers to the Watson/Crick base-pairing interactions between two strands of oligonucleotides within a duplex.
  • overhang refers to a single-stranded terminus of a duplex of base-paired oligonucleotides.
  • the overhang may be one or more bases in length and allows for annealing of a complementary oligonucleotide prior to ligation and extension during polynucleotide assembly.
  • Denaturing conditions or reagents disrupt base-pairing and cause separation of a duplex into single-strands.
  • Denaturing conditions and reagents include heat, basic pH, high salt concentrations and specific denaturants, such as formamide and ammonium hydroxide.
  • Non-denaturing conditions allow base-pairing in duplex structures to persist. Non-denaturing conditions typically include low temperature, neutral pH, low salt concentrations, neutral aqueous buffers, and reagents which do not disrupt hydrogen bonding between nucleobases.
  • ligate refers to the reaction of covalently joining adjacent oligonucleotides through formation of an internucleotide linkage.
  • ligase refers to a class of enzymes and their functions in forming a phosphodiester bond in adjacent oligonucleotides which are annealed to the same oligonucleotide. Particularly efficient ligation takes place when the terminal phosphate of one oligonucleotide and the terminal hydroxyl group of an adjacent second oligonucleotide are annealed together across from their complementary sequences within a double helix, i.e. where the ligation process ligates a "nick” at a ligatable nick site and creates a complementary duplex (Blackburn, 1996).
  • the site between the adjacent oligonucleotides is referred to as the "ligatable nick site", "nick site”, or "nick”, whereby the phosphodiester bond is non-existent, or cleaved.
  • the intervening single-stranded portion between two oligonucleotides in a duplex is referred to as a "gap", consisting of one or more nucleotides.
  • a gap can be eliminated or “filled in” by extension from a 3' terminus of a primer.
  • Primer extension reaction refers to a reaction between a template/primer duplex, 5' triphosphate nucleotides (NTP), and a polymerase which results in the addition of the nucleotide to a 3'-end of the primer such that the added nucleotides are complementary to the corresponding nucleotides of the template nucleic acid.
  • NTP 5' triphosphate nucleotides
  • Label refers to a group attached to an oligonucleotide.
  • the label is capable of conducting a function such as giving a signal for detection of the molecule by such means as fluorescence, chemiluminescence, and electrochemical luminescence (Hermanson, 1996).
  • the label allows for separation or immobilization of the molecule by a specific or non-specific capture method (Andrus, 1995).
  • Primer refers to an oligonucleotide capable of selectively annealing to a specified target nucleic acid and thereafter serving as a point of initiation of a primer extension reaction wherein the primer is extended in a 5' ⁇ 3' direction.
  • 5' ⁇ 3' nuclease activity refers to an enzyme activity that cleaves nucleic acid at phosphodiester bonds. This activity can be either endo (cleaves at internal phosphodiester bonds) or exo (cleaves at the phosphodiester bond closest to either the 5' or 3' terminus of the nucleic acid strand.
  • self-quenching refers to an intermolecular, fluorescence energy transfer effect, e.g. a reporter and quencher are joined on an oligonucleotide in a configuration that permits energy transfer from the reporter to the quencher.
  • oligonucleotide synthesis (Beaucage, 1992; Caruthers, 1983).
  • the phosphoramidite method of oligonucleotide synthesis (Beaucage, 1983; Beaucage, 1992) is the universally favored method of preparing the oligonucleotides used in the invention.
  • the phosphoramidite method is a highly refined chemical operation of cyclical addition of nucleotide monomer units to a chain of DNA growing on a solid-support and is usually practiced using automated, commercially available, synthesizers, which function as microprocessor-controlled, reagent delivery robots, e.g.
  • ABI 391, 392, 394, and 3948 DNA/RNA Synthesizers (Perkin-Elmer Corp) (Caruthers, 1984).
  • the 5' or 3' terminus of an oligonucleotide can be phosphorylated with a phosphoramidite reagent (Horn, 1986) or enzymatically with polynucleotide kinase and ATP (Berger, 1987, p. 438-39).
  • Oligonucleotides may immobilized on solid supports through any one of a variety of well-known covalent linkages or non-covalent interactions.
  • the support is comprised of insoluble materials, preferably having a rigid or semi-rigid character, and may be any shape, e.g. spherical, as in beads, rectangular, irregular particles, resins, gels, microspheres, or substantially flat.
  • it may be desirable to create an array of physically separate synthesis regions on the support with, for example, wells, raised regions, dimples, pins, trenches, rods, pins, inner or outer walls of cylinders, and the like.
  • Preferred support materials include agarose, polyacrylamide, magnetic beads (Stamm, 1995), polystyrene (Andrus, 1993), controlled-pore-glass (Caruthers, 1984), polyacrylate, hydroxethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, or copolymers and grafts of such.
  • Polyethyleneoxy/polystyrene co-polymer is used extensively for small molecule and peptide synthesis and is a particularly preferred solid support of the,present invention (Tentagel, Rapp Polymere, Tubingen, Germany).
  • the hydrophilic nature of the polyethyleneoxy groups promotes rapid kinetics and binding when aqueous solvents are used.
  • Other embodiments of solid-supports include small particles, membranes, frits, non-porous surfaces, addressable arrays, vectors, plasmids, or polynucleotide-immobilizing media.
  • oligonucleotides are attached by covalent bonds, ionic bonds, or other affinity interactions, to chemically reactive functionality on the solid-supports. Oligonucleotides can be attached to solid-supports at their 3', 5', sugar, or nucleobase sites (Goodchild, 1990; Beaucage, 1993). The 3' site for attachment via a linker to the support is preferred due to oligonucleotide synthesis ease and efficiency, and due to the many options available for stable or selectively cleavable linkers (Beaucage, 1992).
  • gram to kilogram scale preparations of immobilized oligonucleotides can be obtained at loading ranges of 1-2000 nmoles oligonucleotide per gram of support, and preferably in a range of 500-1000 nmoles oligonucleotide per gram of support.
  • Immobilization is preferably accomplished by a covalent linkage between the support and the oligonucleotide.
  • the linkage unit, or linker is designed to be stable and facilitate accessibility of the immobilized nucleic acid to its sequence complement.
  • non-covalent linkages such as between biotin and avidin or stepavidin are useful.
  • a typical method for attaching oligonucleotides is coupling a thiol functionalized polystyrene bead with a 3' thiol-oligonucleotide under mild oxidizing conditions to form a disulfide linker.
  • Examples of other functional group linkers include ester, amide, carbamate, urea, sulfonate, ether, and thioester.
  • a 5' or 3' biotinylated oligonucleotide can be immobilized on avidin or strepavidin bound to a support such as glass or SEPHAROSETTM (Pharmacia Biotech).
  • an oligonucleotide can be immobilized to a solid-support.
  • the directionality of the assembled polynucleotide and the component oligonucleotides of the preceding embodiments would thus be reversed, although equally accomodated and efficient.
  • Oligonucleotides are preferably annealed for assembly in aqueous media which promotes Watson/Crick base-pairing, at or near room temperature.
  • 1 mg of support (1 nmole, loaded at 1 ⁇ mole oligonucleotide/gm) is annealed with 5 nmole of each oligonucleotide during each annealing and ligation cycle, in a total volume of 10-50 ⁇ l solution.
  • a ligation reagent effects ligation of a ligatable nick site located between two assembly oligonucleotides.
  • DNA ligase conducts enzymatic ligation upon a ligatable nick site to create an internucleotide phosphodiester bond and create a continuous strand in the immobilized ligation product.
  • Ligation with DNA ligase is highly specific and generally occurs only with perfect complementarity close to the nick site.
  • DNA ligase catalyzes the formation of a phosphodiester bond between the 5' phosphoryl terminus and the 3'-hydroxyl terminus of two, double-stranded oligonucleotides (Wu, 1987; Helfman, 1987; Grossman, 1994).
  • the 5' phosphate group of an assembly oligonucleotide is ligated to the 3' hydroxyl of an adjacent assembly oligonucleotide.
  • the 5' terminus of the ligatable nick site is phosphorylated and the 3' terminus is hydroxyl, although the opposite orientation of 5' hydroxyl and 3' phosphate also leads to efficient ligation by DNA ligase (Sambrook, 1989, p. 5.61).
  • Enzymatic ligation of the assembled polynucleotide on solid-support can be conducted by treating the assembled polynucleotide on solid-support (e.g. 1c., Figure 1) e.g.
  • a ligatable nick site of an assembled polynucleotide can also be chemically ligated with reagents, such as cyanogen bromide and dicyclohexylcarbodiimide, to form an internucleotide phosphate linkage between two adjacent assembled oligonucleotides one of which bears a 5' or 3' phosphate group, annealed to a bridging oligonucleotide (Shabarova, 1991).
  • reagents such as cyanogen bromide and dicyclohexylcarbodiimide
  • the solid-support may be washed under denaturing conditions after each ligation to remove the non-immobilized strands.
  • Preferred denaturants include sodium hydroxide, ammonium hydroxide, formamide, urea, sodium chloride and sodium acetate.
  • a primer complementary to the polynucleotide is annealed to the polynucleotide.
  • a DNA polymerase catalyzes the sequential joining of complementary nucleotides from nucleotide 5'-triphosphates to the 3' terminus of the primer by formation of new internucleotide phosphate bonds. A new complementary strand of DNA is thus extended from the primer.
  • immobilized strands of the assembled polynucleotide may be annealed to one or more bridging oligonucleotides, across from which the nick sites were ligated.
  • the single stranded portions, or "gaps" of the assembled polynucleotide may be filled in by primer extension, followed by ligation of the nick.
  • the 3' terminus within a gap in a duplex can be extended from the 3' terminus of a primer by a DNA polymerase, and 2'-deoxynucleotide-5'-triphosphates, under known conditions (Berger, 1987, p. 91-98).
  • Polymerase enzymes suitable for use in the extension step of the synthesis methods of the invention or for use in the amplification of the polynucleotide by polymerase chain reaction include any that are capable of polymerizing nucleotide triphosphates from a polynucleotide immobilized to a solid-support Preferred polymerase enzymes have high fidelity and processivity. Suitable enzymes include, but are not limited to, DNA Polymerase I, Klenow fragment of DNA Polymerase I, T7 DNA Polymerase, T4 Polymerase, Taq Polymerase, and AMV (or MuLV) Reverse Transcriptase or closely homologous mutants (Sambrook, 1989, p. 5.35-56). More preferably, the enzyme for the extension step and the polymerase chain reaction is Taq Polymerase, or closely homologous mutant.
  • the non-immobilized strand can be removed from the immobilized ligation product under denaturing conditions.
  • Primer extension with poiymerase, a primer, and nucleotide 5' triphosphates can copy the immobilized strand from the priming site.
  • the primer will extend at its 3' hydroxyl toward the 3' terminus of the immobilized strand.
  • Nucleotide 5' triphosphates (NTP) suitable for use in the extension step of the synthesis methods of the invention or for use in the amplification of the polynucleotide by polymerase chain reaction include any that are capable of being polymerized by a polymerase enzyme.
  • Suitable NTPs include both naturally occurring and synthetic nucleotide triphosphates, and are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-ATP, 2-amino-dATP, as well as the ⁇ -thiotriphosphates, 2'-O-methyl-ribonucleotide 5'-triphosphates 2'-fluoro-NTP, and 2'-amino-NTP for all of the above.
  • the nucleotide triphosphates used in the methods of invention are selected from the group consisting of dATP, dCTP, dGTP, TTP, and mixtures thereof.
  • Modified nucleobases can also be used, including but not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.
  • Most of these nucleotide triphosphates are widely available from commercial sources such as Sigma Chemical Co., St. Louis, MO. Nucleotide triphosphates are advantageously used in the methods of the present invention at least because they are generally cheaper than the phosphoramidite nucleoside monomers used in the chemical synthesis of oligonucleotides.
  • fluorescent-labelled dNTP can be added, or substituted for one or more of ATP, GTP, CTP, TTP, to incorporate fluorescent dyes into the double-stranded assembled polynucleotide product.
  • Typical conditions for primer extension can include the addition of the following solution (1-50 ⁇ l) to the assembled polynucleotide on solid support (50-1000 pmole) comprising: primer oligonucleotide (if required), 1 unit DNA polymerase, 80 mM Tris-HCl (pH 8.0), 10 mM dithiothreitol, 4 mM spermidine, 8 mM MgCl 2 , 50 mM NaCl, 160 ⁇ g/ml BSA, 0.02% Triton X-100, and 2 mM each of ATP, GTP, CTP, TTP.
  • an assembly oligonucleotide 10 preferably having a 5' phosphate group 11 , is immobilized, e.g. 1-10 mg, 0.5-20 nmoles, to a solid support 15 through a linker 16 (1a., Figure 1).
  • the immobilized assembly oligonucleotide 20 is suspended in an assembly solvent, e.g. 0.2 M NaCl or KCl and 0-50% formamide.
  • Aqueous assembly solvents which facilitate Watson/Crick base-pairing at or near room temperature are preferred.
  • a bridging oligonucleotide 25 e.g.
  • Assembly oligonucleotide 40 anneals to overhang 35 of the bridging oligonucleotide 25 and adjacent to the immobilized oligonucleotide 10 , creating a ligatable nick site 45 and a second overhang 50 (1c.).
  • a ligating agent e.g. DNA ligase, ATP, and other reagents necessary for ligation, are added to ligate the immobilized assembly oligonucleotide 20 , to the adjacent assembly oligonucleotide 40 to form an immobilized ligation product 60 (1d.).
  • a complement to the immobilized ligation product is then synthesized with DNA polymerase, a primer, nucleotide 5' triphosphates, and other reagents necessary for primer extension to create a double-stranded polynucleotide on the solid-support 65 (1e.).
  • an immobilized assembly oligonucleotide 20 (2a., Figure 2) is suspended in an assembly solvent.
  • a bridging oligonucleotide 25 with a sequence at least partially complementary to the immobilized oligonucleotide, and an assembly oligonucleotide 40 , with a sequence at least partially complementary to the bridging oligonucleotide, are added as a mixture.
  • the bridging oligonucleotide 25 anneals to the immobilized assembly oligonucleotide 20 and the assembly oligonucleotide 40 anneals adjacent to the immobilized assembly oligonucleotide 20 , creating a ligatable nick site 45 and an overhang 50 (2b.). Excess or non-annealed oligonucleotides 25 and 40 , and other impurities may be removed by washing under non-denaturing conditions.
  • a ligating agent e.g. DNA ligase, ATP, and other reagents necessary for ligation are added to ligate the immobilized assembly oligonucleotide 20 to the adjacent assembly oligonucleotide 40 to form an immobilized ligation product 60 (2c.).
  • a complement to the immobilized ligation product is then synthesized with DNA polymerase, a primer, nucleotide 5' triphosphates, and other reagents necessary for primer extension to create a double-stranded polynucleotide on the solid-support 65 (2d.).
  • an immobilized assembly oligonucleotide 20 (3a., Figure 3) is suspended in an assembly solvent. More than two annealing oligonucleotides, e.g. 40a-c , are added as a mixture.
  • the mixture contains one or more bridging oligonucleotides 25a-c which anneal to form gaps 70a-b and one or more assembly oligonucleotides 40a-c that anneal to form ligatable nick sites 45a-c (3b.).
  • Excess or non-annealed oligonucleotides 25 and 40 , and other impurities may be removed by washing under non-denaturing conditions.
  • a ligating agent e.g. DNA ligase, ATP, and other reagents necessary for ligation are added to ligate the nick sites at adjacent assembly oligonucleotides to form an immobilized ligation product 60 (3c.).
  • a complement to the immobilized ligation product is then synthesized with DNA polymerase, a primer, nucleotide 5' triphosphates, and other reagents necessary for primer extension to create a double-stranded polynucleotide on the solid-support 65 (3d.).
  • the steps of sequential annealing of a bridging and an assembly oligonucleotide, as described in section III.1, followed by ligation, and interspersed with washing steps may be repeated up to 100 times or more (4e.).
  • the repetitively annealed and ligated immobilized ligation product 75 is copied with DNA polymerase, a primer, nucleotide 5' triphosphates. and other reagents necessary for extension to create an immobilized double-stranded polynucleotide on the solid-support 65 (4f.).
  • the steps of concurrent annealing of a bridging and an assembly oligonucleotide, as described in section III.2, followed by ligation, and interspersed with washing steps may be repeated up to 100 times or more (5d.).
  • the repetitively annealed and ligated immobilized ligation product 75 is copied with DNA polymerase, a primer, nucleotide 5' triphosphates, and other reagents necessary for extension to create an immobilized double-stranded polynucleotide on the solid-support 65 (5e.).
  • the steps of concurrent annealing of more than one bridging oligonucleotide and more than one assembly oligonucleotide, followed by ligation, and interspersed with washing steps, as described in section III.3, may be repeated up to 100 times or more (6d.).
  • the repetitively annealed and ligated immobilized ligation product 75 is copied with DNA polymerase, a primer, nucleotide 5' triphosphates, and other reagents necessary for extension to create an immobilized double-stranded polynucleotide on the solid-support 65 (6e.).
  • a gene of known DNA sequence and of particular interest is selected for assembly.
  • the size of the gene may range from 50 bp to 5000 bp or more.
  • one strand of the polynucleotide sequence to be synthesized is divided into a contiguous set of assembly oligonucleotide sequences of 20-200 nt, preferably 30-50 nt.
  • Bridging oligonucleotides of 6-40 nt are designed to anneal to assembly oligonucleotides and form the nick sites on the immobilized strand.
  • the extent of complementary overlap in the oligonucleotides forming the duplex regions may be any length so as to provide sufficient specificity and affinity.
  • the complementary overlap will be 5 to 10 nt and may be up to 50 nt.
  • the assembly and bridging oligonucleotides comprising the assembled gene are selected according to the predicted annealing properties, i.e. thermal melting temperature, T m .
  • T m thermal melting temperature
  • the duplex regions resulting from annealing of the oligonucleotides must be stable enough to endure the washing step, and other manipulations, and to undergo efficient ligation.
  • Assembly polynucleotides may contain: (i) nucleotide units such as A, dA, C, dC, G, dG, U, T, dU, 5-methyl-C, 5- methyl-dC, I, dI, 2-amino-A, 2-amino-dA, 5-Br-U, 5-Br-dU, 5-F-U, 5-F-dU, 5-propynyl dC, 5-propynyl-dU, (ii) internucleotide linkages such as phosphodiester, phosphorothioate, N-3-phosphoramidate, and (iii) sugars such as 2'-deoxyribose, 2'-O-methyl-ribonucleotides, 2'-fluoro-ribonucleotides, and 2'-amino-ribonucleotides analogs.
  • nucleotide units such as A, dA, C, dC, G, d
  • oligonucleotide sequence design is (i) avoiding self-complementary hairpin regions, (ii) avoiding poor synthesis efficiency regions, e.g. four or more consecutive G monomers, (iii) rare or poorly expressed codons, and (iv) placement of restriction sites for cleavage and further cloning operations.
  • the entire set of oligonucleotides required to practice the assembly methods of the present invention can thus be designed and synthesized.
  • the immobilized double-stranded polynucleotide sequence may be a conserved, or universal sequence, and not part of the functional gene.
  • the sequence of the immobilized fragment may contain a restriction site cleavable by a restriction enzyme.
  • the immobilized oligonucleotide may be linked to a larger polynucleotide fragment, such as a plasmid or vector.
  • suitable plasmids for the present invention include M13-derived vectors, pUC, and pGEM (Sambrook, 1989, Chapter 1), which can be grown and harvested from large scale bacterial culture (Berger, 1987, p. 145-70) and cut at known restriction sites for assembly of polynucleotides.
  • Immobilized ligation products may be amplified as templates by the polymerase chain reaction (Stamm, 1995). After assembly of, e.g. 50-1000 pmole, immobilized ligation product is complete, PCR reagents may be added as a solution, including DNA polymerase, nucleotide 5' triphosphates, and two primers complementary to (i) the immobilized ligation product and (2) its complement. The temperature may be cycled between the annealing/extension and denaturation temperatures to generate double-stranded polynucleotide copies, in solution, of the immobilized ligation product.
  • Incorporation of fluorescent dyes can generate fluorescent-labelled and detectable polynucleotides.
  • Multiple PCR products of different or the same sizes can be obtained from a single assembled polynucleotide with a plurality of primers, each complementary to different portions of the immobilized ligation product, and selected as pairs on opposing strands.
  • primers defining certain PCR products are labeled with different fluorescent dyes, the multiple PCR products can be spectrally discriminated, thereby detected and quantitated.
  • Multiplex PCR on solid-support is also a convenient, efficient way to handle templates for PCR on solid-support, giving rise to less contamination from adventitious template dispersal and errant amplification.
  • the sequence of the immobilized ligation product can be analyzed by solid-phase Sanger dideoxy DNA sequencing methods.
  • the assays include a self-quenching oligonucleotide probe which is complementary to a portion of the immobilized ligation product.
  • the probe includes a fluorescent reporter dye and quencher arranged to interact through a fluorescence resonance energy transfer (FRET) effect (Clegg, R., 1992).
  • FRET fluorescence resonance energy transfer
  • the quencher can interact with the reporter to alter its light emission, usually resulting in the decreased emission efficiency of the reporter. The efficiency of quenching diminishes with distance from the reporter to the quencher.
  • the probe may be comprised of nucleotides near the 5' terminus which are substantially complementary to the nucleotides near the 3' terminus whereby the unannealed probe exists in a quenched state.
  • the quenching effect is diminished and fluorescence can be detected.
  • the increase in fluorescence of self-complementary, self-quenching probes ("Molecular Beacons") upon hybridization to target polynucleotides is sufficient for sensitive assay results (Tyagi, 1996; Tyagi, 1997).
  • a fluorescence-based, exonuclease assay provides real time measurements of amplification products during PCR (Lee, 1993; Holland, 1991).
  • a self-quenching fluorescence probe complementary to a site of the immobilized ligation product is included in the PCR mixture.
  • the probe anneals to target and is displaced and cleaved by the 5' ⁇ 3' exonuclease activity of the polymerase ( Figure 9).
  • a fluorescent signal is released that is proportional to the amount of assembled polynucleotide present (Livak 1996; Lee, 1993).
  • the exonuclease assay gives direct detection of PCR products derived from amplification of assembled polynucleotides on solid-support with no further sample processing. As PCR proceeds, polymerase cleaves the annealed probe, separating the reporter and quencher, resulting in an increase in fluorescence.
  • Certain preferred embodiments of the present invention include methods for the end-point and real-time measurements of amplification product formed from the immobilized polynucleotide.
  • an end-point mode the fluorescence measurement is performed after amplification of the assembled polynucleotide is complete.
  • a real-time mode fluorescence measurements is performed multiple times during the amplification reaction, e.g., after each thermocycle of a PCR process. The real-time mode is preferred when a quantitative measure of assembled polynucleotide (loading of polynucleotide per gram solid-support) is required.
  • the reporter dye is separated from the quencher dye by at least 12 nucleotides, the reporter dye is attached at the 5' terminus or 3' terminus of the self-quenching fluorescence probe, and the quencher dye is attached at the 5' terminus or 3' terminus (Livak, 1998).
  • the self-quenching probe is designed so as to bring the reporter into close proximity with the quencher so as to permit efficient energy transfer from the reporter to the quencher (Clegg, 1992; Cardullo, 1988; Livak, 1995).
  • the reporter and quencher may also be attached to the 3' terminal nucleotide.
  • the fluorescer and quencher are attached at internal sites on the polynucleotide.
  • the invention also includes embodiments in which one of the two fluorophores is located at an internal site and the other fluorophore is attached to a terminus of the polynucleotide.
  • Dyes suitable as reporters may also be suitable as quenchers.
  • dyes suitable as quenchers may also be suitable as reporters.
  • 6-carboxy-fluorescein (6-FAM) is labelled at the 5' terminus of the probe as the reporter and 6-carboxytetramethylrhodamine (TAMRA) is labelled at the 3' terminus as the quencher such that the TAMRA dye substantially quenches any fluorescent emissions by 6-FAM until cleaved by polymerase.
  • Preferred embodiments of reporter moieties are fluorescein dyes with the general structure and numbering system below, where L is a linker.
  • Preferred embodiments of fluorescein reporter dyes are 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX), and 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE) ( Figure 7).
  • Other embodiments of reporter moieties are cyanine dyes, dansyl derivatives, and the like.
  • quencher moieties are; (i) rhodamine dyes (Bergot, selected from the group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), and tetrapropano-6-carboxyrhodamine (ROX), and (ii) DABSYL, DABCYL, cyanine, anthraquinone, nitrothiazole,and nitroimidazole compounds and the like ( Figure 8).
  • Rhodamine dyes bear the general structure and numbering system below, where L is a linker.
  • Fluorescein and rhodamine derivatives of the present invention may be substituted at one or more of the numbered positions above.
  • the assembled polynucleotide can be released from the solid-support by cleaving the linker by chemical or enzymatic means, or a combination of both.
  • the assembly and bridging oligonucleotides may be chosen to contain a restriction enzyme recognition sequence, typically of 4-8 base pairs in length, then cleavage of the assembled polynucleotide from the solid-support can occur with the appropriate restriction enzyme.
  • cleavage of the sequence, represented by the example below, within 50 pmole of an assembled polynucleotide can be conducted in a mixture of 1 unit of HindIII restriction enzyme, 10 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, 1 mM dithiothreitol, pH 7.9, at 25 °C, in a total volume of 25 ⁇ l.
  • a single-stranded, assembled polynucleotide on solid-support can be cleaved by restriction enzymes by hybridizing an oligonucleotide of 6-40 nt, or longer, to a restriction site of the polynucleotide, followed by treatment with the corresponding restriction enzyme. Cleavage will occur at the double-stranded restriction site, resulting in separation of the polynucleotide from the solid-support. The sticky end of the cleaved polynucleotide will then be ready for ligation and cloning steps.
  • the assembled polynucleotide may contain labile functionality that is cleavable by chemical reagents.
  • L in the figure above may be a trityl group to be cleaved with a weak acid, such as brief treatment at room temperature with acetic acid.
  • L may be a base labile group such as ester or carbamate to be cleaved with ammonium hydroxide, sodium hydroxide, or other aqueous reagents at or about pH 12.
  • the linker L may be a disulfide functional group cleavable by mild reducing agents such as dithiothreitol (Cleland's reagent).
  • the linker L may be a silyl ether functional group cleavable by fluoride ion with reagents such as tetrabutylammonium fluoride.
  • reagents such as tetrabutylammonium fluoride.
  • Typical conditions of an ester linkage of an assembled polynucleotide on solid-support would include treating about 1 mg of support with 100 ⁇ l of concentrated ammonium hydroxide at 25 °C for 6 hours and withdrawing the supernatant to a separate vessel for removal of the ammonia and water under vacuum.
  • a device may be constructed to synthesize a polynucleotide on a solid-support by automating the steps of annealing, ligation, and primer extension in a cyclical manner according to the present invention.
  • Liquid reagents can be delivered from vessels to the solid-supports under microprocessor control according to a program. Applying the methods of the present invention, particularly the enzymatic means of polynucleotide assembly to solid-support chemistry, takes advantage of the convenience and efficiency realized by other chemical, solid-phase biopolymer and small molecule synthesis methods.
  • Temperature control can be realized by immersing the reaction vessels in cooling or heating fluids, or placement in cooling/heating zones, e.g. heating blocks, ovens, chillers.
  • All steps of the assembly process and thermal cycling during PCR can be conducted between 0-100 °C.
  • the heterogeneous reactions of the present invention whereby liquid reagents are delivered to an immobilized reactant on a stationary solid-phase, can exhibit rapid kinetics and high yields while obviating the need for product work-up, isolation, and purification.
  • iterative processes such as monomer additions in assembling biopolymers, is well suited for solid-support synthesis, by manual and automated means.
  • the present invention lends itself to automation of high-throughput, parallel synthesis of genes.
  • Arrays addressable locations on a surface to which reagents, detection elements, or devices can be located, can be utilized with the present invention.
  • the array is a planar surface with locations fixed in a format within a device by which automated means can visit repeatedly for the purposes of (i) conducting chemical or enzymatic reactions, (ii) detecting changes or interactions, or (iii) fixing or mounting for display a multitude of samples.
  • the spatial arrangement of the synthesis array may be a two-dimensional surface addressable by a programmed, robotic automated liquid delivery apparatus.

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Claims (19)

  1. Verfahren zum Synthetisieren eines Polynukleotids auf einem Festträger, wobei das Verfahren die Schritte umfasst:
    a. Anlagerung eines oder mehrerer Brücken-Oligonukleotide und zweier oder mehrerer Assemblierungs-Oligonukleotide, wobei die Brücken-Oligonukleotide 6-40 Nukleotide umfassen, so dass eine ligierbare Nickstelle zwischen benachbarten Assemblierungs-Oligonukleotiden gebildet wird, wobei die Assemblierungs-Oligonukleotide 20-200 Nukleotide umfassen, die Brücken-Oligonukleotide Lücken von einem oder mehreren Nukleotiden in dem nicht-immobilisierten Strang bilden und eines der Assemblierungs-Oligonukleotide an einem Festträger immobilisiert ist;
    b. Ligieren der ligierbaren Nickstellen, wobei dadurch ein immobilisiertes Ligationsprodukt gebildet wird;
    c. Wiederholen der Schritte a. und b. mit 1 bis 100 Zyklen;
    d. Entfernen der Brücken-Oligonukleotide unter denaturierenden Bedingungen;
    e. Anlagerung eines Primers an das immobilisierte Ligationsprodukt und
    f. Verlängerung des Primers mit Polymerase und Nukleotid-5'-Triphosphaten, um ein immobilisiertes doppelsträngiges Polynukleotid zu erzeugen.
  2. Verfahren nach Anspruch 1, wobei der Festträger umfasst ist von kleinen Teilchen, Kügelchen, Membranen, Fritten, nicht-porösen Oberflächen, adressierbaren Anordnungen, Vektoren, Plasmiden oder Polynukleotid-immobilisierenden Medien.
  3. Verfahren nach Anspruch 1 zum Synthetisieren eines Polynukleotids mit einer Länge zwischen 50 und 5000 Nukleotid-Basenpaaren.
  4. Verfahren nach Anspruch 1, wobei der Schritt f. der Verlängerung des Primers, der an das immobilisierte Ligationsprodukt angelagert ist, Nukleotid-5'-Trisphosphate umfasst, ausgewählt aus der Gruppe bestehend aus:
    ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-Methyl-CTP, 5-Methyl-dCTP, ITP, dITP, 2-Amino-ATP, 2-Amino-dATP, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-Propynyl-dCTP, 5-Propynyl-dUTP und
    die korrespondierenden α-Thiotriphosphate, 2'-O-Methylribonukleotidtriphosphate, 2'-Fluor-NTP, 2'-Amino-NTP-Analoge und mit Fluoreszenz markierte NTP.
  5. Verfahren nach Anspruch 4, wobei die fluoreszierenden Markierungen an die N-9- oder C-8-Position des Purins oder Deazapurins und die C-5-Position des Pyrimidins gebunden sind und ausgewählt sind aus der Gruppe bestehend aus FAM, TET, HEX oder JOE.
  6. Verfahren nach Anspruch 1, wobei die Assemblierungs- und Brücken-Oligonukleotide Nukleotideinheiten enthalten, ausgewählt aus der Gruppe bestehend aus:
    A, dA, C, dC, G, dG, U, T, dU, 5-Methyl-C, 5-Methyl-dC, I, dI, 2-Amino-A, 2-Amino-dA, 5-Br-U, 5-Br-dU, 5-F-U, 5-F-U, 5-F-dU, 5-Propynyl-dC, 5-Propynyl-dU und
    die korrespondierenden Phosphorothioate, N-3-Phosphoramidate, 2'-O-Methylribonukleotide, 2'-Fluorribonukleotide und 2'-Aminoribonukleotid-Analoge.
  7. Verfahren nach Anspruch 1, weiter umfassend den Schritt des periodischen Waschens unter nicht-denaturierenden Bedingungen nach den Schritten a. und b.
  8. Verfahren nach Anspruch 1, weiter umfassend die Schritte des Waschens des Trägers unter denaturierenden Bedingungen nach jedem Ligationsschritt b.
  9. Verfahren nach Anspruch 8, wobei die Denaturierungsmittel ausgewählt sind aus der Gruppe bestehend aus Natriumhydroxid, Ammoniumhydroxid, Formamid, Harnstoff, Natriumchlorid und Natriumacetat.
  10. Verfahren nach Anspruch 1, wobei die Polynukleotide durch chemische Spaltung vom Träger gespalten werden.
  11. Verfahren nach Anspruch 1, wobei die Polynukleotide durch enzymatische Spaltung vom Träger gespalten werden.
  12. Verfahren nach Anspruch 1, weiter umfassend den Schritt der Anlagerung einer selbst-löschenden Fluoreszenzsonde, die komplementär zu dem immobilisierten Polynukleotid ist.
  13. Verfahren nach Anspruch 12, wobei die selbst-löschende Fluoreszenzsonde umfasst wird von Nukleotiden in der Nähe des 5'-Terminus, die im Wesentlichen komplementär sind zu den Nukleotiden in der Nähe des 3'-Terminus, wobei die nichtangelagerte Sonde in einem gelöschten Zustand vorliegt.
  14. Verfahren nach Anspruch 12, wobei die Anlagerung der selbst-löschenden Fluoreszenzsonde an das immobilisierte doppelsträngige Polynukleotid durch Fluoreszenznachweis gemessen wird.
  15. Verfahren nach Anspruch 1, wobei der Schritt f. der Verlängerung des Primers, der an das immobilisierte Ligationsprodukt angelagert ist, eine Polymerase-Kettenreaktion ist, umfassend:
    eine thermostabile Nukleinsäure-Polymerase, die 5'→3'-Nukleaseaktivität hat, wobei der Primer komplementär ist zu dem immobilisierten Ligationsprodukt, ein zweiter Primer, der komplementär ist zu dem Gegenstück des immobilisierten Ligationsprodukts, 5'-Nukleotidtriphosphate und eine selbst-löschende Fluoreszenzsonde;
    wobei die Sonde in mindestens einer Einzelstrangkonformation vorliegt, wenn sie nicht an das Polynukleotid angelagert ist, wobei ein Quencher die Fluoreszenz eines Reporters und mindestens eine Konformation löscht, wenn er an das immobilisierte Ligationsprodukt angelagert ist, wobei die Fluoreszenz des Reporters nicht gelöscht wird, und die Schritte
    Anlagerung der Primer an das immobilisierte Ligationsprodukt;
    Amplifizieren des immobilisierten Ligationsprodukts durch PCR, wobei Ziel-Polynukleotid-Amplifikationsprodukte erzeugt werden.
  16. Verfahren nach Anspruch 15, wobei die Nukleinsäure-Polymerase die selbst-löschende Fluoreszenzsonde während der Amplifikation verdaut, um den Reporter von dem Quencher zu trennen.
  17. Verfahren nach Anspruch 15, wobei die Ziel-Polynukleotid-Amplifikationsprodukte durch Fluoreszenznachweis gemessen werden.
  18. Verfahren nach Anspruch 15, wobei die Ziel-Polynukleotid-Amplifikationsprodukte durch Endpunktanalyse gemessen und quantifiziert werden.
  19. Verfahren nach Anspruch 15, wobei die Ziel-Polynukleotid-Amplifikationsprodukte durch Echtzeitanalyse gemessen und quantifiziert werden.
EP99945166A 1998-11-12 1999-08-24 Ligationsassemblierung und detektion von polynucleotiden auf einem festträger Expired - Lifetime EP1129215B1 (de)

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AU770217B2 (en) 2004-02-19
JP2002531071A (ja) 2002-09-24
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